Project Summary/Abstract Homeostatic regulation of nutrients is critical for maintaining health, but high nutrient intake can tip the balance and cause pathological nutrient regulation, such as glucose and lipid dysregulation, a common occurrence in obesity that leads to diabetes. The incidence of obesity and its associated comorbidities are rapidly increasing, but there is a limited understanding of the molecular and cellular mechanisms underlying obesity-induced pathological progression. Acquiring this understanding as well as what endogenous protective mechanisms exist to prevent disease progression at the molecular level can inform future strategies for novel treatments of the disease. Sestrin2 is a stress-induced protein that is required for glucose and lipid homeostasis in high-fat diet (HFD) fed mice. However, it is unknown whether forced upregulation of Sestrin2 can be used to treat diabetic pathologies during obesity and insulin resistance. Preliminary results demonstrated that liver-specific overexpression (OE) of Sestrin2 in HFD mice almost completely normalized blood glucose homeostasis and liver fat levels while minimally affecting previously established Sestrin2 signaling pathways. Interestingly, Sestrin2 OE strongly activated AKT, even without insulin stimulation. AKT is a major effector of insulin signaling, and its activation promotes glucose uptake, glycogenesis, and inhibits gluconeogenesis. AKT is also known to promote hepatic lipogenesis, hepatomegaly and carcinogenesis, but these deleterious outcomes were not induced by Sestrin2 OE. Therefore, it is plausible that Sestrin2 promotes the beneficial activities of AKT for glucose homeostasis without affecting its deleterious activities in fat accumulation and carcinogenesis. Considering the strong metabolic effects of Sestrin2 and the important metabolic functions of AKT, this project aims to 1) determine the molecular mechanism for how Sestrin2 activates AKT, and 2) determine the downstream pathways activated by Sestrin2-induced AKT activation. We will use a combination of biochemical and cell-based assays, gene editing, mouse models, and computational approaches to accomplish the project goals. The proposed research will provide insight into a novel mechanism of nutrient regulation for insulin signaling and resistance under conditions of hypernutrition and provide new therapeutic targets for alleviating diabetic nutrient dysregulation pathologies.